ABSTRACT: To develop strategies to block parenteral transmission of HIV, it is critical to understand the mechanisms that initiate HIV infection and viral spread in vivo. Though for 25 years, the field has largely focused on cell-free mechanisms of viral spread to model and understand the disease, a compelling case can be made for the spread of HIV through direct contact between infected and uninfected cells. Our recent studies have focused on the cell biology of intercellular adhesive structures, called virological synapses, that enhance viral spread between cells. Using an innovative strategy to visualize viral transmission through synapses, we engineered a fluorescent, infectious molecular clone of HIV. At virological synapses, we uncovered dynamic movements of assembling virus particles in infected T cells, as well as a novel endocytic process that accompanies synapse formation and infection. The complexity of the cellular processes and their high efficiency suggests that this may be the dominant paradigm for HIV dissemination. We will test the hypothesis that the virological synapse represents the fundamental unit of transmission in vivo. Blocking the synapse, either with antibodies or with other antagonists, may be the key to breaking the cycle of transmission. For this avant garde project, we will exploit recently developed small humanized animal models to examine the sequence of events that occurs during parenteral HIV transmission. We will test the relative efficiency of cell-free versus cell-associated routes, and track viral dissemination in the first minutes and hours after viral challenge. To reveal the cell-cell and cell-virus interactions at an organismal level, we will employ in vivo imaging approaches to track the movement of infected or virus-carrying cells within humanized mice. We will reveal a sequence of interactions between infected cells/virus and uninfected cells. The results will provide new strategies to interfere with viral transmission and dissemination.